Cartridge and analyzer for fluid analysis

ABSTRACT

A fluidic cartridge and methods of operation are described. The fluidic cartridge includes a substrate having a plurality of contact pads designed to electrically couple with an analyzer, a semiconductor chip having a sensor array, and a reference electrode. The fluidic cartridge includes a first fluidic channel having an inlet and coupled to a second fluidic channel, the second fluidic channel being aligned such that the sensor array and the reference electrode are disposed within the second fluidic channel. A first plug is disposed at the first inlet. The first plug includes a compliant material configured to be punctured by a capillary without leaking fluid through the first plug.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.15/406,066, filed on Jan. 13, 2017, which claims priority of U.S.Provisional Patent Application No. 62/372,596, filed Aug. 9, 2016, eachof which is incorporated by reference in its entirety.

BACKGROUND

Biosensors are devices for sensing and detecting biomolecules andoperate on the basis of electronic, electrochemical, optical, andmechanical detection principles. Biosensors that include transistors aresensors that electrically sense charges, photons, and mechanicalproperties of bio-entities or biomolecules. The detection can beperformed by detecting the bio-entities or biomolecules themselves, orthrough interaction and reaction between specified reactants andbio-entities/biomolecules. Such biosensors can be manufactured usingsemiconductor processes, can quickly convert electric signals, and canbe easily applied to integrated circuits (ICs) and MEMS.

The interaction of the biological sample itself and the biosensor can bea challenge. Typically, a fluid containing the biological sample ispipetted directly over the sensing portion of the biosensor. This methodleads to a large portion of the fluid sample not being used, and is timeconsuming to manually load each sensing area.

BRIEF DESCRIPTION OF THE DRAWINGS

Aspects of the present disclosure are best understood from the followingdetailed description when read with the accompanying figures. It isnoted that, in accordance with the standard practice in the industry,various features are not drawn to scale. In fact, the dimensions of thevarious features may be arbitrarily increased or reduced for clarity ofdiscussion.

FIG. 1 is a diagram illustrating components of an exemplary biosensingcartridge.

FIG. 2 is a cross-sectional view of an exemplary dual-gate back-sidesensing FET Sensor.

FIG. 3 is a circuit diagram of a plurality of FET Sensors configured inan exemplary addressable array.

FIG. 4 is a circuit diagram of an exemplary addressable array of dualgate FET Sensors and heaters.

FIG. 5 is a cross-sectional view of an exemplary dual gate back-sidesensing FET Sensor configured as a pH sensor.

FIG. 6A illustrates an example of the binding of ions to a receptorlayer.

FIG. 6B illustrates a change in threshold voltage in an exemplary FETSensor based on pH.

FIG. 7 is a floor plan diagram of an exemplary biosensor chip.

FIG. 8 shows a series of cross-sectional views illustrating afabrication process for mounting an exemplary biosensor chip to a handlelayer.

FIG. 9 is a top view of the handle layer having the exemplary biosensorchip mounted to a substrate.

FIG. 10 is a schematic of an exemplary fluidic cartridge having andintegrated biosensor chip.

FIG. 11 is a schematic of some of the fluid channels in the exemplaryfluidic cartridge.

FIG. 12 is a schematic of the exemplary fluidic cartridge coupled to ananalyzer.

FIG. 13 is a flow diagram of an exemplary method of using the fluidiccartridge.

FIG. 14 is a cross-sectional view of an exemplary dual gate back-sidesensing bioFET detecting DNA.

FIG. 15A illustrates the binding mechanics of DNA on a receptor surface.

FIG. 15B illustrates a change in threshold voltage for the exemplarydual gate back-side sensing bioFET based on matched analyte binding.

FIG. 16 is a cross-sectional view of an exemplary dual gate back-sidesensing bioFET having antibodies immobilized on its sensing layer.

FIG. 17 illustrates the binding mechanics of antigens and antibodies ona receptor surface.

DETAILED DESCRIPTION

The following disclosure provides many different embodiments, orexamples, for implementing different features of the provided subjectmatter. Specific examples of components and arrangements are describedbelow to simplify the present disclosure. These are, of course, merelyexamples and are not intended to be limiting. For example, the formationof a first feature over a second feature in the description that followsmay include embodiments in which the first and second features areformed in direct contact, and may also include embodiments in whichadditional features may be formed and/or disposed between the first andsecond features, such that the first and second features may not be indirect contact. In addition, the present disclosure may repeat referencenumerals and/or letters in the various examples. This does not in itselfdictate a relationship between the various embodiments and/orconfigurations discussed.

Further, spatially relative terms, such as “beneath,” “below,” “lower,”“above,” “upper” and the like, may be used herein for ease ofdescription to describe one element or feature's relationship to anotherelement(s) or feature(s) as illustrated in the figures. The spatiallyrelative terms are intended to encompass different orientations of thedevice in use or operation in addition to the orientation depicted inthe figures. The apparatus may be otherwise oriented (e.g., rotated 90degrees or at other orientations) and the spatially relative descriptorsused herein may likewise be interpreted accordingly.

Terminology

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this disclosure belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of embodiments in accordance with thedisclosure; the methods, devices, and materials are now described. Allpatents and publications mentioned herein are incorporated herein byreference for the purpose of describing and disclosing the materials andmethodologies which are reported in the publications which might be usedin connection with the invention.

The acronym “FET,” as used herein, refers to a field effect transistor.A very common type of FET is referred to as a metal oxide semiconductorfield effect transistor (MOSFET). Historically, MOSFETs have been planarstructures built in and on the planar surface of a substrate such as asemiconductor wafer. But recent advances in semiconductor manufacturinghave resulted in three-dimensional, of fin-based, MOSFET structures.

The term “bioFET” refers to a FET that includes a layer of immobilizedcapture reagents that act as surface receptors to detect the presence ofa target analyte of biological origin. A bioFET is a field-effect sensorwith a semiconductor transducer, according to an embodiment. Oneadvantage of bioFETs is the prospect of label-free operation.Specifically, bioFETs enable the avoidance of costly and time-consuminglabeling operations such as the labeling of an analyte with, forinstance, fluorescent or radioactive probes. One specific type of bioFETdescribed herein is a dual-gate back-side sensing bioFET. The analytesfor detection by a BioFET will normally be of biological origin, suchas—without limitation—proteins, carbohydrates, lipids, tissue fragmentsor portions thereof. However, in a more general sense a BioFET is partof a broader genus of FET sensors that may also detect any chemicalcompound (known in the art as a ChemFET), or any other element,including ions, such as protons or metallic ions (known in the art as anISFET). This invention is meant to apply to all types of FET-basedsensors (“FET Sensor”). One specific type of FET Sensor herein is aDual-Gate Back Side Sensing FET Sensor (“DG BSS FET Sensor”).

“S/D” refers to the source/drain junctions that form two of the fourterminals of a FET.

The expression “high-k” refers to a high dielectric constant. In thefield of semiconductor device structures and manufacturing processes,high-k refers to a dielectric constant that is greater than thedielectric constant of SiO₂ (i.e., greater than 3.9).

The term “analysis” generally refers to a process or step involvingphysical, chemical, biochemical, or biological analysis that includes,but is not limited to, characterization, testing, measurement,optimization, separation, synthesis, addition, filtration, dissolution,or mixing.

The term “assay” generally refers to a process or step involving theanalysis of a chemical or a target analyte and includes, but is notlimited to, cell-based assays, biochemical assays, high-throughputassays and screening, diagnostic assays, pH determination, nucleic acidhybridization assays, polymerase activity assays, nucleic acid andprotein sequencing, immunoassays (e.g., antibody-antigen binding assays,ELISAs, and iqPCR), bisulfite methylation assays for detectingmethylation pattern of genes, protein assays, protein binding assays(e.g., protein-protein, protein-nucleic acid, and protein-ligand bindingassays), enzymatic assays, coupled enzymatic assays, kineticmeasurements (e.g., kinetics of protein folding and enzymatic reactionkinetics), enzyme inhibitor and activator screening, chemiluminescenceand electrochemiluminescence assays, fluorescent assays, fluorescencepolarization and anisotropy assays, absorbance and colorimetric assays(e.g., Bradford assay, Lowry assay, Hartree-Lowry assay, Biuret assay,and BCA assay), chemical assays (e.g., for the detection ofenvironmental pollutants and contaminants, nanoparticles, or polymers),and drug discovery assays. The apparatus, systems, and methods describedherein may use or adopt one or more of these assays to be used with anyof the FET Sensor described designs.

The term “liquid biopsy” generally refers to a biopsy sample obtainedfrom a subject's bodily fluid as compared to a subject's tissue sample.The ability to perform assays using a body fluid sample is oftentimesmore desirable than using a tissue sample. The less invasive approachusing a body fluid sample has wide ranging implications in terms ofpatient welfare, the ability to conduct longitudinal disease monitoring,and the ability to obtain expression profiles even when tissue cells arenot easily accessible, e.g., in the prostate gland. Assays used todetect target analytes in liquid biopsy samples include, but are notlimited to, those described above. As a non-limiting example, acirculating tumor cell (CTC) assay can be conducted on a liquid biopsysample.

For example, a capture reagent (e.g., an antibody) immobilized on a FETSensor may be used for detection of a target analyte (e.g., a tumor cellmarker) in a liquid biopsy sample using a CTC assay. CTCs are cells thathave shed into the vasculature from a tumor and circulate, e.g., in thebloodstream. Generally CTCs are present in circulation in extremely lowconcentrations. To assay the CTCs, CTCs are enriched from patient bloodor plasma by various techniques known in the art. CTCs may be stainedfor specific markers using methods known in the art including, but notlimited to, cytometry (e.g., flow cytometry)-based methods and IHC-basedmethods. For the apparatus, systems, and methods described herein, CTCsmay be captured or detected using a capture reagent or the nucleicacids, proteins, or other cellular milieu from the CTCs may be targetedas target analytes for binding to or detection by a capture reagent.

When a target analyte is detected on or from a CTC, e.g., an increase intarget analyte expressing or containing CTCs may help identify thesubject as having a cancer that is likely to respond to a specifictherapy (e.g., one associated with a target analyte) or allow foroptimization of a therapeutic regimen with, e.g., an antibody to thetarget analyte. CTC measurement and quantitation can provide informationon, e.g., the stage of tumor, response to therapy, disease progression,or a combination thereof. The information obtained from detecting thetarget analyte on the CTC can be used, e.g., as a prognostic,predictive, or pharmacodynamic biomarker. In addition, CTCs assays for aliquid biopsy sample may be used either alone or in combination withadditional tumor marker analysis of solid biopsy samples.

The term “identification” generally refers to the process of determiningthe identity of a target analyte based on its binding to a capturereagent whose identity is known.

The term “measurement” generally refers to the process of determiningthe amount, quantity, quality, or property of a target analyte based onits binding to a capture reagent.

The term “quantitation” generally refers to the process of determiningthe quantity or concentration of a target analyte based on its bindingto a capture reagent.

The term “detection” generally refers to the process of determining thepresence or absence of a target analyte based on its binding to acapture reagent. Detection includes but is not limited toidentification, measurement, and quantitation.

The term “chemical” refers to a substance, compound, mixture, solution,emulsion, dispersion, molecule, ion, dimer, macromolecule such as apolymer or protein, biomolecule, precipitate, crystal, chemical moietyor group, particle, nanoparticle, reagent, reaction product, solvent, orfluid any one of which may exist in the solid, liquid, or gaseous state,and which is typically the subject of an analysis.

The term “reaction” refers to a physical, chemical, biochemical, orbiological transformation that involves at least one chemical and thatgenerally involves (in the case of chemical, biochemical, and biologicaltransformations) the breaking or formation of one or more bonds such ascovalent, noncovalent, van der Waals, hydrogen, or ionic bonds. The termincludes typical chemical reactions such as synthesis reactions,neutralization reactions, decomposition reactions; displacementreactions, reduction-oxidation reactions, precipitation,crystallization, combustion reactions, and polymerization reactions, aswell as covalent and noncovalent binding, phase change, color change,phase formation, crystallization, dissolution, light emission, changesof light absorption or emissive properties, temperature change or heatabsorption or emission, conformational change, and folding or unfoldingof a macromolecule such as a protein.

“Capture reagent” as used herein, is a molecule or compound capable ofbinding the target analyte or target reagent, which can be directly orindirectly attached to a substantially solid material. The capture agentcan be a chemical, and specifically any substance for which there existsa naturally occurring target analyte (e.g., an antibody, polypeptide,DNA, RNA, cell, virus, etc.) or for which a target analyte can beprepared, and the capture reagent can bind to one or more targetanalytes in an assay.

“Target analyte” as used herein, is the substance to be detected in thetest sample using the present invention. The target analyte can be achemical, and specifically any substance for which there exists anaturally occurring capture reagent (e.g., an antibody, polypeptide,DNA, RNA, cell, virus, etc.) or for which a capture reagent can beprepared, and the target analyte can bind to one or more capturereagents in an assay. “Target analyte” also includes any antigenicsubstances, antibodies, and combinations thereof. The target analyte caninclude a protein, a peptide, an amino acid, a carbohydrate, a hormone,a steroid, a vitamin, a drug including those administered fortherapeutic purposes as well as those administered for illicit purposes,a bacterium, a virus, and metabolites of or antibodies to any of theabove substances.

“Test sample” as used herein, means the composition, solution,substance, gas, or liquid containing the target analyte to be detectedand assayed using the present invention. The test sample can containother components besides the target analyte, can have the physicalattributes of a liquid, or a gas, and can be of any size or volume,including for example, a moving stream of liquid or gas. The test samplecan contain any substances other than the target analyte as long as theother substances do not interfere with the binding of the target analytewith the capture reagent or the specific binding of the first bindingmember to the second binding member. Examples of test samples include,but are not limited to naturally-occurring and non-naturally occurringsamples or combinations thereof. Naturally-occurring test samples can besynthetic or synthesized. Naturally-occurring test samples include bodyor bodily fluids isolated from anywhere in or on the body of a subject,including but not limited to, blood, plasma, serum, urine, saliva orsputum, spinal fluid, cerebrospinal fluid, pleural fluid, nippleaspirates, lymph fluid, fluid of the respiratory, intestinal, andgenitourinary tracts, tear fluid, saliva, breast milk, fluid from thelymphatic system, semen, cerebrospinal fluid, intra-organ system fluid,ascitic fluid, tumor cyst fluid, amniotic fluid and combinationsthereof, and environmental samples such as ground water or waste water,soil extracts, air, and pesticide residues or food-related samples.

Detected substances can include, e.g., nucleic acids (including DNA andRNA), hormones, different pathogens (including a biological agent thatcauses disease or illness to its host, such as a virus (e.g., H7N9 orHIV), a protozoan (e.g., Plasmodium-causing malaria), or a bacteria(e.g., E. coli or Mycobacterium tuberculosis)), proteins, antibodies,various drugs or therapeutics or other chemical or biologic orsubstances, including hydrogen or other ions, non-ionic molecules orcompounds, polysaccharides, small chemical compounds such as chemicalcombinatorial library members, and the like. Detected or determinedparameters may include but are not limited to, e.g., pH changes, lactosechanges, changing concentration, particles per unit time where a fluidflows over the device for a period of time to detect particles, e.g.,particles that are sparse, and other parameters.

As used herein, the term “immobilized,” when used with respect to, e.g.,a capture reagent, includes substantially attaching the capture reagentat a molecular level to a surface. For example, a capture reagent may beimmobilized to a surface of the substrate material using adsorptiontechniques including non-covalent interactions (e.g., electrostaticforces, van der Waals, and dehydration of hydrophobic interfaces) andcovalent binding techniques where functional groups or linkersfacilitate attaching the capture reagent to the surface. Immobilizing acapture reagent to a surface of a substrate material may be based uponthe properties of the substrate surface, the medium carrying the capturereagent, and the properties of the capture reagent. In some cases, asubstrate surface may be first modified to have functional groups boundto the surface. The functional groups may then bind to biomolecules orbiological or chemical substances to immobilize them thereon.

The term “nucleic acid” generally refers to a set of nucleotidesconnected to each other via phosphodiester bond and refers to anaturally occurring nucleic acid to which a naturally occurringnucleotide existing in nature is connected, such as DNA comprisingdeoxyribonucleotides having any of adenine, guanine, cytosine, andthymine connected to each other and/or RNA comprising ribonucleotideshaving any of adenine, guanine, cytosine, and uracil connected to eachother. In addition, non-naturally occurring nucleotides andnon-naturally occurring nucleic acids are within the scope of thenucleic acid of the present invention. Examples include peptide nucleicacids (PNA), peptide nucleic acids with phosphate groups (PHONA),bridged nucleic acids/locked nucleic acids (BNA/LNA), and morpholinonucleic acids. Further examples include chemically-modified nucleicacids and nucleic acid analogues, such as methylphosphonate DNA/RNA,phosphorothioate DNA/RNA, phosphoramidate DNA/RNA, and 2′-O-methylDNA/RNA. Nucleic acids include those that may be modified. For example,a phosphoric acid group, a sugar, and/or a base in a nucleic acid may belabeled as necessary. Any substances for nucleic acid labeling known inthe art can be used for labeling. Examples thereof include but are notlimited to radioactive isotopes (e.g., 32P, 3H, and 14C), DIG, biotin,fluorescent dyes (e.g., FITC, Texas, cy3, cy5, cy7, FAM, HEX, VIC, JOE,Rox, TET, Bodipy493, NBD, and TAMRA), and luminescent substances (e.g.,acridinium ester).

Aptamer as used herein refers to oligonucleic acids or peptide moleculesthat bind to a specific target molecule. The concept of usingsingle-stranded nucleic acids (aptamers) as affinity molecules forprotein binding was initially disclosed in 1990 (Ellington and Szostak1990, 1992; Tuerk and Gold 1990), and is based on the ability of shortsequences to fold, in the presence of a target, into unique,three-dimensional structures that bind the target with high affinity andspecificity. Eugene W. M Ng et al., 2006, discloses that aptamers areoligonucleotide ligands that are selected for high-affinity binding tomolecular targets.

The term “antibody” as used herein refers to a polypeptide of theimmunoglobulin family that is capable of binding a corresponding antigennon-covalently, reversibly, and in a specific manner. For example, anaturally occurring IgG antibody is a tetramer comprising at least twoheavy (H) chains and two light (L) chains inter-connected by disulfidebonds. Each heavy chain is comprised of a heavy chain variable region(abbreviated herein as VH) and a heavy chain constant region. The heavychain constant region is comprised of three domains, CH1 CH2 and CH3.Each light chain is comprised of a light chain variable region(abbreviated herein as VL) and a light chain constant region. The lightchain constant region is comprised of one domain, CL. The VH and VLregions can be further subdivided into regions of hypervariability,termed complementarity determining regions (CDR), interspersed withregions that are more conserved, termed framework regions (FR). Each VHand VL is composed of three CDRs and four FRs arranged fromamino-terminus to carboxy-terminus in the following order: FR1, CDR1,FR2, CDR2, FR3, CDR3, and FR4. The three CDRs constitute about 15-20% ofthe variable domains. The variable regions of the heavy and light chainscontain a binding domain that interacts with an antigen. The constantregions of the antibodies may mediate the binding of the immunoglobulinto host tissues or factors, including various cells of the immune system(e.g., effector cells) and the first component (C1q) of the classicalcomplement system. (Kuby, Immunology, 4th ed., Chapter 4. W.H. Freeman &Co., New York, 2000).

The term “antibody” includes, but is not limited to, monoclonalantibodies, human antibodies, humanized antibodies, chimeric antibodies,and anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Idantibodies to antibodies of the invention). The antibodies can be of anyisotype/class (e.g., IgG, IgE, IgM, IgD, IgA and IgY), or subclass(e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2).

The term “polymer” means any substance or compound that is composed oftwo or more building blocks (‘mers’) that are repetitively linked toeach other. For example, a “dimer” is a compound in which two buildingblocks have been joined together. Polymers include both condensation andaddition polymers. Typical examples of condensation polymers includepolyamide, polyester, protein, wool, silk, polyurethane, cellulose, andpolysiloxane. Examples of addition polymers are polyethylene,polyisobutylene, polyacrylonitrile, poly(vinyl chloride), andpolystyrene. Other examples include polymers having enhanced electricalor optical properties (e.g., a nonlinear optical property) such aselectroconductive or photorefractive polymers. Polymers include bothlinear and branched polymers.

Overview of Biosensing Cartridge

FIG. 1 illustrates an overview of various components that are integratedtogether to form an exemplary biosensing cartridge 102. Biosensingcartridge 102 may include a plurality of fluidic channels that areconfigured to control fluid flow both towards and away from a sensinglocation where the presence of a target analyte can be detected.

In this illustrative embodiment, biosensing cartridge 102 includes anarray of FET Sensors 104. FET Sensors 104 make up the transducercomponent of biosensing cartridge 102. FET Sensors 104 may be arrangedin an array and individually addressed to detect binding events at thesurface of the FET Sensor sensing layer. In one embodiment, FET Sensors104 include dual gate back-side FET Sensors. In alternative embodimentsother types of FET Sensor-based sensors may be used.

Biosensing cartridge 102 includes a biological interface 106. Biologicalinterface 106 may be coupled to dual gate back-side sensing FET Sensors104 to facilitate binding reactions at the surface of dual gateback-side sensing FET Sensors 104, which can be then be detected.Various types of biomolecules may form a part of biological interface106, such as DNA or RNA aptamers and antibodies, to name a few examples.Further details regarding the biological interface, and its associatedchemistry and biology mechanics, will be discussed in detail herein.

Biosensing cartridge 102 includes various levels of chip packaging 108in order to integrate a dual gate back-side sensing FET Sensor chip intoa fluidic environment. Biosensing cartridge 102 also includes a fluidiccomponent 110 having microfluidic channels to manage delivery of liquidsto FET Sensors 104. Fluidic component 110 also incorporates fluidicinlets for interfacing with fluids that are delivered from outside ofbiosensing cartridge 102.

The integration of various components in biosensing cartridge 102 yieldsa compact and portable platform that can be used for a multitude ofvarious biosensing applications. The use of FET Sensors with theintegrated fluidics component produces accurate results while using lowsample volumes. Additionally, biosensing cartridge 102 may be configuredto be operated in a fully autonomous way by an analyzer, and thendisposed of after use.

The description herein is split into four major sections to describecomponents of biosensing cartridge 102 in further detail. The firstsection will describe the arrangement and fabrication of dual gateback-side bioFET sensors 104. The second section will describe thepackaging process. The third section will describe fluidic component110, and further describe the interaction between biosensing cartridge102 and an analyzer. The final section will provides details regardingthe biology and the various biosensing applications using dual gateback-side FET Sensors 104.

Dual Gate Back-Side FET Sensors

Dual gate back-side FET Sensors utilize semiconductor manufacturingtechniques and biological capture reagents to form sensitive and easilyarrayed sensors. While conventional MOSFETs have a single gate electrodethat is connected to a single electrical node, the dual gate back-sidesensing FET Sensor has two gate electrodes each of which is connected toa different electrical node. A first one of the two gate electrodes isreferred to herein as the front-side gate and the second one of the twogate electrodes is referred to herein as the back-side gate. Both thefront-side gate and the back-side gate are configured such that, inoperation, each one may be electrically charged and/or discharged andthereby each influences the electric field between the source/drainterminals of the dual gate back-side sensing FET Sensor. The front-sidegate is electrically conductive, separated from a channel region by afront-side gate dielectric, and configured to be charged and dischargedby an electrical circuit to which it is coupled. The back-side gate istypically separated from the channel region by a back-side gatedielectric, and includes a biofunctionalized sensing layer disposed onthe back-side gate dielectric. The amount of electric charge on theback-side gate is a function of whether a biorecognition reaction hasoccurred. In the typical operation of dual gate back-side sensing FETSensors, the front-side gate is charged to a voltage within apredetermined range of voltages. The voltage on the front-side gatedetermines a corresponding conductivity of the FET Sensor's channelregion. A relatively small amount of change to the electric charge onthe back-side gate changes the conductivity of the channel region. It isthis change in conductivity that indicates a biorecognition reaction.

One advantage of FET Sensors is the prospect of label-free operation.Specifically, FET Sensors enable the avoidance of costly andtime-consuming labeling operations such as the labeling of an analytewith, for instance, fluorescent or radioactive probes.

Referring to FIG. 2, illustrated is an exemplary dual gate back-sidesensing FET sensor 200. Dual gate back-side sensing FET sensor 200includes a control gate 202 formed over substrate 214 and separatedtherefrom by an intervening dielectric 215 disposed on substrate 214.Substrate 214 further includes a source region 204, a drain region 206,and a channel region 208 between source region 204 and drain region 206.In an embodiment, substrate 214 has a thickness between about 100 nm andabout 130 nm. Gate 202, source region 204, drain region 206, and channelregion 208 may be formed using suitable CMOS process technology. Gate202, source region 204, drain region 206, and channel region 208 form aFET. An isolation layer 210 is disposed on the opposing side ofsubstrate 214 from gate 202. In one embodiment, isolation layer 210 hasa thickness of about 1 μm. In this disclosure the side of substrate 214over which gate 202 is disposed is referred to as the “front-side” ofsubstrate 214. Similarly, the side of substrate 214 on which isolationlayer 210 is disposed is referred to as the “back-side.”

An opening 212 is provided in isolation layer 210. Opening 212 may besubstantially aligned with gate 202. In other embodiments, opening 212is larger than gate 202 and may extend over multiple dual gate back-sidesensing FET Sensors. An interface layer (not shown) may be disposed inopening 212 on the surface of channel region 208. The interface layermay be operable to provide an interface for positioning and immobilizingone or more receptors for detection of biomolecules or bio-entities.Further details regarding the interface layer are provided herein.

Dual gate back-side sensing FET sensor 200 includes electrical contactsto drain region 206 (Vd 216), source region 204 (Vs 218), gate structure202 (front-side gate 220), and/or active region 208 (e.g., back-sidegate 222). It should be noted that back-side gate 222 does not need tophysically contact substrate 214 or any interface layer over substrate214. Thus, while a conventional FET uses a gate contact to controlconductance of the semiconductor between the source and drain (e.g., thechannel), dual gate back-side sensing FET sensor 200 allows receptorsformed on the opposing side of the FET device to control theconductance, while gate structure 202 provides another gate to controlthe conductance. Therefore, dual gate back-side sensing FET sensor 200may be used to detect one or more specific biomolecules or bio-entitiesin the environment around and/or in opening 212, as discussed in moredetail using various examples herein.

Dual gate back-side sensing FET sensor 200 may be connected toadditional passive components such as resistors, capacitors, inductors,and/or fuses; and other active components, including P-channel fieldeffect transistors (PFETs), N-channel field effect transistors (NFETs),metal-oxide-semiconductor field effect transistors (MOSFETs), highvoltage transistors, and/or high frequency transistors; other suitablecomponents; and/or combinations thereof. It is further understood thatadditional features can be added in dual gate back-side sensing FETsensor 200, and some of the features described can be replaced oreliminated, for additional embodiments of dual gate back-side sensingFET sensor 200. Further details regarding example fabrication proceduresof dual gate back-side sensing FET sensor 200 may be found in co-ownedU.S. Patent Publication No. 2013/0200438 and U.S. Patent Publication No.2014/0252421.

Referring to FIG. 3, illustrated is a schematic of an exemplaryaddressable array 300 of FET Sensors 304 connected to bit lines 306 andword lines 308. It is noted that the terms bit lines and word lines areused herein to indicate similarities to array construction in memorydevices, however, there is no implication that memory devices or astorage array necessarily be included in the array. Addressable array300 may have similarities to that employed in other semiconductordevices such as dynamic random access memory (DRAM) arrays. For example,dual gate back-side sensing FET Sensor 200, described above withreference to FIG. 2, may be formed in a position that a capacitor wouldbe found in a DRAM array. Schematic 300 is exemplary only and one wouldrecognize other configurations are possible.

FET Sensors 304 may each be substantially similar to dual gate back-sidesensing FET Sensor 200. FETs 302 are configured to provide connectionbetween a drain terminal of FET Sensor 304 and bit line 306. In this wayFETs 302 are analogous to access transistors in a DRAM array. In thisexemplary embodiment, FET Sensors 304 is a dual gate back-side sensingFET Sensor and includes a sensing gate provided by a receptor materialdisposed on a dielectric layer overlying a FET active region disposed ata reaction site, and a control gate provided by a gate electrode (e.g.,polysilicon) disposed on a dielectric layer overlying the FET activeregion.

Schematic 300 shows an array formation that may be advantageous indetecting small signal changes provided by minimal biomolecules orbio-entities introduced to FET Sensors 304. The arrayed format using bitlines 306 and word lines 308 allows for a decreased number ofinput/output pads. Amplifiers may be used to enhance the signal strengthto improve the detection ability of the device having the circuitarrangement of schematic 300. In an embodiment, when particular wordlines 308 and bit lines 306 are asserted, the corresponding accesstransistors 302 will be turned on (e.g., like a switch.) When the gateof the associated FET Sensor 304 (e.g., such as back-side gate 222 ofthe dual gate back-side sensing FET sensor 200) has its charge affectedby the bio-molecule presence, FET Sensor 304 will transfer electrons andinduce the field effect charging of the device, thereby modulating thecurrent (e.g., I_(ds)). The change of the current (e.g., I_(ds)) orthreshold voltage (V_(t)) can serve to indicate detection of therelevant biomolecules or bio-entities. Thus, the device having schematic300 can achieve a biosensor application including application withdifferential sensing for enhanced sensitivity.

Referring to FIG. 4, an exemplary layout 400 is presented. Exemplarylayout 400 includes access transistor 302 and FET Sensor 304 arranged asan array 401 of individually addressable pixels 402. Array 401 mayinclude any number of pixels 402. For example, array 401 may include128×128 pixels. Other arrangements may include 256×256 pixels ornon-square arrays such as 128×256 pixels.

Each pixel 402 includes access transistor 302 and dual gate back-sidesensing FET Sensor 304 along with other components that may include oneor more heaters 408 and a temperature sensor 410. In this example,access transistor 302 is an n-channel FET. An n-channel FET 412 may alsoact as an access transistor for temperature sensor 410. In thisillustrative example, the gates of FETs 302 and 412 are coupled incommon, though this is not required. Each pixel 402 (and its associatedcomponents) may be individually addressed using column decoder 404 androw decoder 406. In one example, each pixel 402 has a size of about 10micrometers by about 10 micrometers. In another example, each pixel 402has a size of about 5 micrometers by about 5 micrometers, or has a sizeof about 2 micrometers by about 2 micrometers.

Column decoder 406 and row decoder 404 may be used to determine theON/OFF state of n-channel FETs 302 and 412. Turning on n-channel FET 302provides a current to an S/D region of dual gate back-side sensing FETSensor 304. When these devices are ON, a current I_(ds) flows throughFET Sensor 304 and may be measured.

Heater 408 may be used to locally increase a temperature around a dualgate back-side sensing FET Sensor 304. Heater 408 may be constructedusing any known technique, such as forming a metal pattern with a highcurrent running through it. Heater 408 may also be a thermoelectricheater/cooler, like a Peltier device. Heater 408 may be used duringcertain biological tests, such as to denature DNA or RNA, or to providea more ideal binding environment for certain biomolecules. Temperaturesensor 410 may be used to measure the local temperature around dual gateback-side sensing FET Sensor 304. In one embodiment, a control loop maybe created to control the temperature using heater 408 and the feedbackreceived from temperature sensor 410. In another embodiment, heater 408may be a thermoelectric heater/cooler that allows for local activecooling of the components within pixel 402.

Referring to FIG. 5, a cross section of an example dual gate back-sidesensing FET Sensor 500 is provided. The dual gate back-side sensing FETSensor 500 is one implementation of dual gate back-side sensing FETSensor 200, thus previously described elements from FIG. 2 are labeledwith element numbers from FIG. 2 and their descriptions are not repeatedhere. Dual gate back-side sensing FET Sensor 500 includes gate 202,source region 204, drain region 206, and channel region 208, wheresource region 204 and drain region 206 are formed within substrate 214.Gate 202, source region 204, drain region 206, and channel region 208form a FET. It should be noted that the various components of FIG. 5 arenot intended to be drawn to scale and are exaggerated for visualconvenience, as would be understood by a person skilled in the relevantart.

In an exemplary embodiment, dual gate back-side sensing FET Sensor 500is coupled to various layers of metal interconnects 502 that makeelectrical connection with the various doped regions and other devicesformed within substrate 214. Metal interconnects 502 may be manufacturedusing fabrication processes well known to a person skilled in therelevant art.

Dual gate back-side FET Sensor 500 may include a body region 504separate from source region 204 and drain region 206. Body region 504may be used to bias the carrier concentration in active region 208between source region 204 and drain region 206. As such, a negativevoltage bias may be applied to body region 504 to improve thesensitivity of dual gate back-side FET Sensor 500. In one embodiment,body region 504 is electrically connected with source region 204. Inanother embodiment, body region 504 is electrically grounded.

Dual gate back-side FET Sensor 500 may be coupled to additionalcircuitry 506 fabricated within substrate 214. Circuitry 506 may includeany number of MOSFET devices, resistors, capacitors, or inductors toform circuitry to aid in the operation of dual gate back-side sensingFET Sensor 500. For example, column decoder 406 and row decoder 404 maybe formed in circuitry 506. Circuitry 506 may include any amplifiers,analog to digital converters (ADCs), digital to analog converters(DACs), voltage generators, logic circuitry and DRAM memory, to name afew examples. All or some of the components of additional circuitry 506may be integrated in the same substrate 214 as dual gate back-side FETSensor 500. It should be understood that many FET sensors, eachsubstantially similar to dual gate back-side FET Sensor 500, may beintegrated on substrate 214 and coupled to additional circuitry 506. Inanother example, all or some of the components of additional circuitry506 are provided on another semiconductor substrate separate fromsubstrate 214. In yet another example, some components of additionalcircuitry 506 are integrated in the same substrate 214 as dual gateback-side FET Sensor 500, and some components of additional circuitry506 are provided on another semiconductor substrate separate fromsubstrate 214.

Still referring to the illustrative example of FIG. 5, dual gateback-side sensing FET Sensor 500 includes an interface layer 508deposited over isolation layer 210 and within the opening over channelregion 208. In one embodiment, interface layer 508 has a thicknessbetween about 20 Å and about 40 Å. Interface layer 508 may be a high-Kdielectric material, such as hafnium silicate, hafnium oxide, zirconiumoxide, aluminum oxide, tantalum pentoxide, hafnium dioxide-alumina(HfO₂—Al₂O₃) alloy, or any combinations thereof. Interface layer 508 mayact as a support for the attachment of capture reagents as will bediscussed in more detail later in the section directed to biologicalsensing.

An example operation of dual gate back-side FET Sensor 500 acting as apH sensor will now be described. Briefly, a fluid gate 510 is used toprovide the electrical contact to the “second gate” of the dual gateback-side sensing FET Sensor. A solution 512 having a given pH isprovided over the reaction site of dual gate back-side sensing FETSensor 500, and fluid gate 510 is placed within solution 512. The pH ofthe solution is generally related to the concentration of hydrogen ions[H⁺] in the solution. The accumulation of the ions near the surface ofinterface layer 508 above channel region 208 will affect the formationof the inversion layer within channel region 208 that forms theconductive pathway between source region 204 and drain region 206. Thiscan be measured by the change in the conductivity of the FET Sensor. Inone embodiment, fluid gate 510 is used as the gate of the transistorduring sensing while gate 202 remains floating. In another embodiment,fluid gate 510 is used as the gate of the transistor during sensingwhile gate 202 is biased at a given potential. For example, gate 202 maybe biased at a potential between −2V and 2V depending on theapplication, while fluid gate 510 is swept between a range of voltages.In another embodiment, fluid gate 510 is biased at a given potential (orgrounded) while gate 202 is used as the gate of the transistor (e.g.,its voltage is swept across a range of potentials) during sensing. Fluidgate 510 may be formed from platinum or may be formed from any othercommonly used material(s) for reference electrodes in electrochemicalanalysis. The most common reference electrode is the Ag/AgCl electrode,having a stable potential value of about 0.230 V.

FIG. 6A shows ions in solution binding to a surface of interface layer508. A top-most atomic layer of interface layer 508 is depicted as thevarious dangling [O⁻], [OH], and [OH₂ ⁺] bonds. As the ions accumulateon the surface, the total surface charge affects the threshold voltageof the transistor. As used herein, the threshold voltage is the minimumpotential between the gate and the source of a FET Sensor that isrequired to form a conductive path of minority carriers between thesource and the drain of the FET sensor. The total charge also directlyrelates to pH of the solution, as a higher accumulation of positivecharge signifies a low pH while a higher accumulation of negative chargesignifies a high pH. FIG. 6B illustrates the change in threshold voltagethat results due to different pH values in an n-channel FET Sensor. Ascan be observed in the figure, a 59 mV increase in threshold voltageroughly signifies an increase of one in the pH of the solution. In otherwords, one pH change results in total surface charge equivalent of 59 mVwhen measured as the voltage required to turn on the transistor.

Chip Packaging

Referring to FIG. 7, an exemplary floor plan for a semiconductor chip702 is shown. Chip 702 includes sensor array 704, an optional referenceelectrode 706, analog circuitry 708, and I/O pads 716. Chip 702 may besilicon, gallium arsenide, or indium phosphide to name a few examples.Chip 702 may have dimensions of about 3 mm by about 2.5 mm.

Sensor array 704 represents the array of dual gate back-side sensing FETSensors such as those illustrated above in FIGS. 2 and 5. The array maybe arranged as a row-column matrix of pixels as illustrated, forexample, in FIG. 4. The various FET Sensors in sensor array 704 may befunctionalized with the same or different capture reagents to performbiosensing for various analytes.

Reference electrode 706 may be patterned on the same chip 702 thatincludes sensor array 704. Reference electrode 706 may be roughlyaligned with sensor array 704 along an X or Y direction, such that afluidic channel may be placed over both sensor array 704 and referenceelectrode 706. In another embodiment, reference electrode 706 isprovided elsewhere off of chip 702.

Reference electrode 706 may comprise any material having a relativelystable potential. Example reference electrode materials include platinumor Ag/AgCl. Fabricating an Ag/AgCl electrode on a substrate surface iswell known in the art as described, for example, by Moschou et al.,“Surface and Electrical Characterization of Ag/AgCl Pseudo-ReferenceElectrodes Manufactured with Commercially Available PCB Technologies,”Sensors, vol. 15(8), 2015, pp. 18102-18113.

Analog circuitry 708 may include circuitry related to the operation ofsensor array 704. As such, analog circuitry 708 may be configured toprovide signals to, and measure signals from, sensor array 704, whileinterfacing with various I/O pads 716. In one embodiment, analogcircuitry 708 includes a serial peripheral interface (SPI) 712, andsensor array circuitry 714. In this embodiment, a spacing between sensorarray 704 and sensor array circuitry 714 is no shorter than about 135micrometers.

SPI 712 may be a serial interface circuit to facilitate datatransmission between sensor array circuitry 714 and an analyzer unitdescribed in more detail below. The general operation of a SPI would bewell understood to a person skilled in the relevant art. Sensor arraycircuitry 714 may include any number of reference voltage generators,operational amplifiers, low pass filters, ADCs, and DACs to providesignals to, and receive signals from, sensor array 704.

In one example, a biasing reference voltage may be generated usingsensor array circuitry 714 to provide a negative voltage bias around−0.24 volts to the body region of a given FET Sensor or set of FETSensors in sensor array 704. A tunable voltage may also be provided tothe fluid gate of a given FET Sensor or set of FET Sensors in sensorarray 704 when performing the sensing.

When measuring signals (such as Ids) received from a given FET Sensor ora set of FET Sensors in sensor array 704, sensor array circuitry 714 mayreceive the measured signals and pass them through a trans-impedanceamplifier, i.e., a current-to-voltage converter, followed by one or moreadditional amplification stages, low pass filters, and ultimately anADC, before the resulting signal is output to an I/O pad 716. Noise mayalso be reduced from the measured signal by subtracting a background ACsignal from the measured signal before the measured signal is amplified.A temperature signal (received from one or more temperature sensors insensor array 704) may also be amplified, filtered, and passed through anADC before being output to an I/O pad 716.

In various embodiments a plurality of I/O pads 716 may be patternedalong the periphery of chip 702. Many more I/O pads may be provided thanactual inputs and outputs used by the various components of chip 702. Inone embodiment, wire bonding techniques may be used to couple variousI/O pads 716 to another substrate or package bonded to chip 702. In oneparticular embodiment, 32 I/O pads may be patterned around the peripheryof chip 702. The size of a given I/O pad 716 may be about 80 micrometersby about 70 micrometers, and the pitch between I/O pads 716 may be about150 micrometers. A spacing between sensor array 704 and a closest I/Opad 716 may be no shorter than about 400 micrometers while a spacingbetween I/O pads 716 and an outermost edge of chip 702 may be no shorterthan about 177.5 micrometers.

Referring to FIG. 8, an exemplary packaging scheme is illustrated forchip 702. Chip 702 with its I/O pads 716 is bonded to a carrier layer802. Carrier layer 802 may be another semiconductor substrate, such as asilicon substrate. In another example, carrier layer 802 is aninsulator, such as a hard plastic material. Chip 702 may be bound tocarrier layer 802 using any known binding techniques, such as by usingsolder or an adhesive.

In one embodiment, carrier layer 802 includes a plurality ofthrough-holes filled with a conductive material 804. Conductive material804 may be any metal such as, but not limited to, tin, copper, aluminum,gold, or any alloy thereof. Conductive material 804 may include a solderbump or solder ball at a bottom surface 805 of carrier layer 802. Thesolder may extend beyond surface 805.

The chip package also includes a first insulating layer 806 that abutsthe sides of chip 702, according to an embodiment. First insulatinglayer 806 may also be a plastic material or resin that fills the areasaround chip 702 and can aid in securing chip 702 in place. In anexemplary embodiment, first insulating layer 806 includes through-holesthat are also filled with conductive plugs 808. Conductive plug 808 maybe the same material as conductive material 804. Conductive plugs 808are substantially aligned over corresponding areas of conductivematerial 804 such that an ohmic contact is formed between conductiveplugs 808 and conductive material 804.

Once chip 702 has been secured to carrier layer 802, and has firstinsulating layer 806 around it, electrical connections 812 may be madebetween I/O pads 716 and conductive plugs 808. Electrical connections812 may be formed using wire-bonding techniques as would be understoodto a person skilled in the relevant art. In another example, electricalconnections 812 are formed using lithographic patterning techniques topattern a conductive trace to electrically connect I/O pads 716 withcorresponding conductive plugs 808. Once electrical connections 812 areformed, a second insulating layer 810 may be deposited to protectelectrical connections 812 from the environment. Second insulating layer810 may be the same material as first insulating layer 806. Secondinsulating layer 810 may be a resin material that flows aroundelectrical connections 812 and then hardens to form a protective shell.An opening 814 is formed within second insulating layer 810 to create apathway towards the sensor array present on chip 702. In an embodimentwhere a reference electrode is also patterned on chip 702, then opening814 would create a pathway towards the sensor array and the referenceelectrode.

A final chip package 816 includes chip 702 bound to carrier layer 802and electrically connected to various conductive solder points or metalpads on bottom surface 805 of carrier layer 802. Chip 702 is alsoprotected from the environment via first insulating layer 806 and secondinsulating layer 810. Chip package 816 may be more easily handled andcoupled to a larger substrate, such as a printed circuit board (PCB). Insome embodiments, chip package 816 may be coupled to one or more heatsinks to provide a more efficient heat dissipation path from chip 702into either the surrounding air or into whatever substrate chip package816 is attached to. In other embodiments, chip package 816 may becoupled to a Peltier device to provide thermoelectric heating and/orcooling.

Referring to the illustrative embodiment of FIG. 9, chip package 816 isbonded with a substrate 902. Substrate 902 may be a PCB that includesconductive contact pads to make electrical contact with the solder orconductive pads on the bottom surface of carrier layer 802. A flip-chipbonding technique may be performed to bond chip package 816 onto thesurface of substrate 902. Briefly, the solder or conductive pads alongthe bottom surface of carrier layer 802 are aligned to correspondingconductive pads patterned on substrate 902, and are bonded together tophysically attach chip package 816 to substrate 902 and to electricallycouple the I/O pads from chip 702 to conductive traces present onsubstrate 902. The conductive traces on substrate 902 may terminate inedge connectors 908.

One or more edge connectors 908 may provide electrical connection tochip 702. One or more other edge connectors 908 may provide electricalconnection to a reference electrode 906 patterned on a surface ofsubstrate 902. Using reference electrode 906 may eliminate the need forproviding a reference electrode on chip 702. Each of the one or moreedge connectors 908 may be patterned using a metal such as, but notlimited, copper, gold, or aluminum. Reference electrode 906 may befabricated using similar techniques as those discussed above forreference electrode 706 on chip 702.

The dimensions of exemplary chip package 816 may be between about one totwo centimeters by one to two centimeters or smaller while thedimensions of substrate 902 may be between three to four centimeters bythree to four centimeters or smaller.

Opening 814 is illustrated over chip 702, exposing at least the sensorarray of chip 702. In an exemplary embodiment, opening 814 is roughlyaligned with reference electrode 906 along an X or Y direction, suchthat a fluidic channel may be placed over both opening 814 and referenceelectrode 906.

Fluidic Design

Referring to FIG. 10, a schematic of an exemplary fluidic cartridge 1000is provided. The schematic illustrates a top-down view of cartridge1000, and it should be noted that not all elements shown are on the samehorizontal plane. Also, the specific dimensions and scale of the variousfluidic channels are purposefully not drawn to scale for improvedvisualization. Cartridge 1000 includes a housing 1002. Housing 1002 maybe formed from any plastic material, such as polymethyl methacrylate(PMMA), using injection molding, casting, or 3-D printing techniques, toname a few examples. Housing 1002 may be formed from more than onesegment that connects together either mechanically or through the use ofan adhesive. In one embodiment, the various fluidic channels andchambers are molded within one or more components of housing 1002. Inanother embodiment, the various fluidic channels and chambers are formedfrom a different molded polymer material, such as polydimethylsiloxane(PDMS). The overall dimensions of housing 1002 may be between about 4centimeters to about 7 centimeters by about 4 centimeters to about 7centimeters. As technology advances, housing 1002 may become evensmaller. In an embodiment, substrate 902 having packaged chip 802 isdisposed within housing 1002. In one example, only a portion ofsubstrate 902 is enclosed within housing 1002, while edge connectors 908are exposed outside of housing 1002.

The fluidic design of exemplary housing 1002 includes at least a firstchannel 1004, a second channel 1006, and a third channel 1008. Each offirst channel 1004 and second channel 1006 includes a correspondingfluid inlet 1010 a and 1010 b, respectively. The fluid inlets provideareas to inject fluid into cartridge 1000 from outside of cartridge1000. The fluid inlets may also provide areas to expel fluid fromcartridge 1000 to outside of cartridge 1000. Third channel 1008 may bealigned over packaged chip 802 bonded to substrate 902. In oneembodiment, opening 814 over the sensor array is substantially withinthird channel 1008. Reference electrode 906 patterned on substrate 902is also aligned to be within third channel 1008, according to anembodiment.

Each of first channel 1004, second channel 1006, and third channel 1008may have channel widths between about one millimeter and threemillimeters. Channel height may be around 1 millimeter. In anotherembodiment, one or more of first channel 1004, second channel 1006, andthird channel 1008 are microfluidic channels having width and heightdimensions less than 1 mm. Each of first channel 1004, second channel1006, and third channel 1008 may have a rectangular, square, orsemi-circular cross-section.

In some embodiments, one or more of first channel 1004 and secondchannel 1006 connect with third channel 1008. In this way, fluid flowingthrough first channel 1004 will flow eventually through third channel1008, and similarly fluid flowing through second channel 1006 will floweventually through third channel 1008. In some embodiments, thirdchannel 1008 eventually flows into a waste chamber 1016 that collectsall fluids flowing through cartridge 1000. Waste chamber 1016 mayinclude a vent (not shown) to the atmosphere to avoid backpressurebuilding up within the fluidic system.

In some embodiments, each inlet 1010 a and 1010 b includes a plug 1012 aand 1012 b, respectively. Plug 1012 a/1012 b may be a soft, compliantmaterial that fits snuggly within inlet 1010 a/1010 b to seal the inletsfrom any fluid leakage. Plug 1012 a/1012 b may be a polymer material,such as polytetrafluoroethylene (PTFE), or cork. Plug 1012 a/1012 b mayseal inlet 1010 a/1010 b while allowing a capillary to puncture throughplug 1012 a/1012 b without compromising the fluidic seal. The capillarymay be a needle-like tube, such as a syringe needle. The capillary maycomprise a hard, rigid material such as a metal or hard plastic. Thecoupling of capillaries to cartridge 1000 will be described in moredetail later when discussing the coupling of cartridge 1000 with ananalyzer.

Cartridge 1000 includes a sample inlet 1014 arranged to introduce asample into either first channel 1004 (as shown in FIG. 10) or secondchannel 1006. In one example, a blood sample may be placed into thefluidic system via sample inlet 1014. Once the sample has beenintroduced, sample inlet 1014 may be sealed using a cap or any othersimilar structure to provide a leak-proof seal around sample inlet 1014.In the illustrated channel arrangement of FIG. 10, fluid flowing throughfirst channel 1004 from inlet 1010 a will mix with a sample introducedvia sample inlet 1014 and the mixture will flow over opening 814 andreference electrode 906 in third channel 1008. Once the sample has beendelivered to the sensor array exposed via opening 814, the interactionbetween the biomolecules can occur and the FET Sensor sensors may beused to detect the presence of, or measure the concentration of,particular analytes in the sample. The fluid may be moved along andbetween the various channels using pressure driven flow. The pressuremay be caused by a syringe forcing liquid or air through cartridge 1000,or by pressurized air pushing against the liquid, to name a fewexamples. Other examples of techniques for transporting liquid throughcartridge 1000 include electro-wetting or using an on-chip peristalticpump. In some embodiments, fluid mixing may occur within cartridge 1000using any one of various on-chip mixing methods known in the art. Thedimensions of the fluid channels of cartridge 1000 may be large enoughthat some fluid mixing occurs due to turbulent flow of the liquid as itmoves through the channel. It should be understood that the location ofsample inlet 1014 can vary. For example, sample inlet 1014 may belocated directly over opening 814 such that a sample introduced intosample inlet 1014 is also introduced over the sensor array exposed viaopening 814.

Once substrate 902 has been integrated into housing 1002, the sensorarray accessed via opening 814 may be functionalized with variouscapture reagents, according to an embodiment. This process may involveflowing a liquid buffer comprising the capture reagents through thirdchannel 1008 such that the capture reagents have the opportunity to bindto the various FET sensors in the sensor array. In another example, thecapture reagents are disposed directly over opening 814 when sampleinlet 1014 is positioned over opening 814. After the capture reagentshave been immobilized, sample inlet 1014 may be sealed such thatcartridge 1000 may be stored until it is ready to perform a biologicalsensing test. The capture reagents may remain within their initialbuffer solution, or a fresh buffer solution may be introduced topreserve the capture reagents while cartridge 1000 awaits testing.Examples of different capture reagents and tests performed with thecapture reagents are provided herein.

Referring to FIG. 11, another design for the various fluidic channels ofcartridge 1000 is illustrated. In this design, a first channel 1104having a first inlet 1102 a and a second channel 1106 having an inlet1102 b converge at an area having a sample inlet 1110. A third channel1108, having opening 814 aligned within it, connects with first channel1104 and second channel 1106 at sample inlet 1110. Opening 814 providesa pathway down to a chip to expose at least the sensor array on the chipto the fluid in third channel 1108. Fluid that flows from either firstchannel 1104 or second channel 1106 through third channel 1108 isultimately collected within waste chamber 1112. Fluid may be directedtowards waste chamber 1112 based on the geometry of the variouschannels, or by using valves to close off certain channels. Sample inlet1110 may also be located over opening 814.

One or more of first channel 1104, second channel 1106, and thirdchannel 1108 may include a bubble trap 1114. Bubble trap 1114 mayrepresent an area of the fluidic channel having a suddenly greatercross-section (or a higher “ceiling”) such that any air present withinthe solution can rise into the extra space created at bubble trap 1114.Other bubble trap designs may be utilized as well as would be understoodby a person having skill in the relevant art. Removal of air bubblesfrom the solution before it reaches the sensor array beneath opening 814may be important to ensure accurate sensing results.

Referring to FIG. 12, cartridge 1000 is illustrated being coupled to ananalyzer 1200 for performing the biological sensing. Cartridge 1000 maybe brought into physical contact with analyzer 1200 by, for example,pressing cartridge 1000 against a receiving port of analyzer 1200. Thereceiving port of analyzer 1200 may include electrical pads to formohmic contacts to some or all of edge connectors 908. An edge ofsubstrate 902 may fit snuggly into a receiving port of analyzer 1200such that edge connectors 908 press against corresponding conductivepads of analyzer 1200. Other methods of assembling cartridge 1000 andanalyzer 1200 include snapping them together, plugging one into theother, among others. Analyzer 1200 may be small enough to be easilyportable and may fit into the palm of an adult human hand.

In some embodiments, analyzer 1200 includes at least a first syringe1202 a and a second syringe 1202 b. Each of first syringe 1202 a andsecond syringe 1202 b may include buffers or other fluids used duringthe operation of cartridge 1000. Syringes 1202 a/1202 b each include aneedle 1204 a/1204 b that may be aligned to extend into space away froma remaining portion of analyzer 1200. In some embodiments, needle 1204a/1204 b may be aligned such that pressing cartridge 1000 against areceiving port of analyzer 1200 causes needle 1204 a/1204 b to puncturethrough corresponding plug 1012 a/1012 b and into inlet 1010 a/1010 b.In this embodiment, needle 1204 a/1204 b is one example of a capillarythat punctures through corresponding plug 1012 a/1012 b. Thus, aleak-proof seal is created to transfer solution from each syringe 1202a/1202 b into the corresponding inlet 1010 a/1010 b of cartridge 1000.It should be understood that although this description describes onlytwo syringes aligned with two input ports, any number of syringes andfluidic input ports may be used, including an example where only onesyringe is used to couple with a single inlet. Each syringe 1202 a/1202b may be preloaded with solution for use in various tests. In anotherembodiment, each syringe 1202 a/1202 b may be easily removed andreplaced with a different syringe by a user.

Each syringe 1202 a/1202 b may have its associated plunger controlledvia a corresponding actuator 1206 a/1206 b. Examples of actuator 1206a/1206 b include a stepper motor or an induction motor. The speed atwhich actuators 1206 a/1206 b depress the plungers of syringes 1202a/1202 b will directly affect the flow rate of the solution within thefluidic channels of cartridge 1000. Actuator 1206 a/1206 b may becontrolled via motor control module 1208 a/1208 b. Motor control module1208 a/1208 b includes the circuitry required to generate voltages forcontrolling the speed and operation of actuator 1206 a/1206 b, as wouldbe understood by a person skilled in the relevant art.

All electrical connections made to edge connectors 908 of cartridge 1000may be routed to sensing electronics 1210. Sensing electronics 1210 mayinclude any number of discrete circuits, integrated circuits, anddiscrete analog circuit components that are designed to both provide andreceive numerous different electrical signals between sensingelectronics 1210 and edge connectors 908. For example, sensingelectronics 1210 may be configured to provide power, ground, and clocksignals to edge connectors 908, which may be subsequently used to powerand operate the sensor array and other electronics on chip 702. Sensingelectronics 1210 may also provide various voltage bias levels foractivating the gates of particular FET Sensors within the sensor array.Sensing electronics 1210 may receive signals that represent draincurrents measured from particular FET Sensors, and signals thatrepresent outputs from temperature sensors on chip 702. Sensingelectronics 1210 may store this received data in a memory, or may usethe received data to alter the voltage bias levels, or to change anamount of heat generated by heaters on chip 702. Generally, sensingelectronics 1210 control all signaling related to the biosensingperformed by the sensor array of cartridge 1000.

In some embodiments, analyzer 1200 also includes a processor 1212 thatcontrols the functions and timing of each of the other modules ofanalyzer 1200, such as motor control module 1208 a/1208 b and sensingelectronics 1210. Processor 1212 may be any type of central processingunit (CPU) or microcontroller and may be programmable by a user toperform certain functions related to the operation of analyzer 1200.Processor 1212 may be configured to analyze signals received fromsensing electronics 1210 to determine a concentration level of a givenanalyte from the sample in cartridge 1000. Data related to thedetermined concentration levels may be stored in a memory of analyzer1200. In another embodiment, sensing electronics 1210 determines aconcentration level of a given analyte from the sample in cartridge1000, and is further configured to store data related to the determinedconcentration levels in a memory of analyzer 1200.

In some embodiments, analyzer 1200 includes a communication module 1214that is designed to communicate data to an external processing device.Processor 1212 may be electrically coupled with communication module1214 to control data transfer. The communication may be wired orwireless. Examples of wired communication include data transfer via anetwork cable or a universal serial bus (USB) cable. Wirelesscommunication may include radio RF transmission, Bluetooth, WiFi, 3G, or4G. Communication module 1214 may also be designed to receive data fromthe external processing device. For example, a program for how tooperate the various components of analyzer 1200 may be transmitted tocommunication module 1214 and executed by processor 1212. Communicationmodule 1214 may include any number of well-known hardware elements tofacilitate analog and/or digital data transmission and reception.

After a biosensing test has been performed, cartridge 1000 may beremoved from analyzer 1200 and discarded. Additionally, syringes 1202a/1202 b may be removed from analyzer 1200 and discarded. Thus, allreagents remain contained within either cartridge 1000 or syringes 1202a/1202 b and no contamination of any other part of analyzer 1200 occurs.In this way, a single analyzer 1200 may be reused to test any number ofadditional cartridges, where each cartridge may be individuallyfunctionalized with different capture reagents to perform a differentbiosensing test.

In another embodiment, syringes 1202 a/1202 b are integrated oncartridge 1000, and the coupling between cartridge 1000 and analyzer1200 aligns the associated plungers of syringes 1202 a/1202 b withactuators 1206 a/1206 b on analyzer 1200. In this embodiment, analyzer1200 is completely free of any reagent-carrying containers.

In another embodiment, cartridge 1000 includes one or more capillariesthat are punctured through corresponding plug 1012 a/1012 b. In thisembodiment, when coupling occurs between cartridge 1000 and analyzer1200, the capillaries fluidically couple with the remainder of syringes1202 a/1202 b in analyzer 1200. After a biosensing test has beenperformed, cartridge 1000 along with its capillaries may be removed fromanalyzer 1200 and discarded.

Referring to FIG. 13, an example method 1300 is presented. Method 1300may be performed by analyzer 1200 after cartridge 1000 has been coupledto analyzer 1200. Other operations relating to fluid handling andelectrical measurement not illustrated in method 1300 may be performedeither before, between, or after the illustrated operations of method1300. The various operations of method 1300 may be performed in adifferent order than the one illustrated. In an embodiment, method 1300is performed after capture reagents have already been immobilized withincartridge 1000.

At block 1302, a first solution flows through a first channel of acartridge. The first solution may enter the cartridge via an inletcoupled to the first channel. The first solution may be provided by asyringe having its needle puncturing through a plug disposed at theinlet of the first channel. The first solution may include a buffersolution to provide a stable pH environment.

At block 1304, the dual gate back-side sensing FET Sensors of the sensorarray are calibrated in the first solution. The calibration may beperformed to measure a noise or background signal of the various FETSensors. This measurement may be stored and later subtracted from themeasured signal when detecting biomolecules to try and reduce the noiseand achieve a clearer detection signal. The first solution must bepresent over the sensor array and the reference electrode patternedwithin the main detection channel to perform the calibration. In someembodiments, the first solution is not flowing during the calibrationmeasurement. In some embodiments, the calibration measurement representsthe baseline threshold voltage for the FET sensors.

At block 1306, a sample is input into the fluidic network of thecartridge via a sample inlet. The sample may be any liquid sample,including a blood sample. In some embodiments, the sample is asemi-solid sample that dissociates within solution. After the sample hasbeen input via the sample inlet, the sample inlet may be sealed by usinga cap or other similar structure.

At block 1308, a second solution flows through a second channel of thecartridge. The second solution may be the same solution as the firstsolution. The second solution may cross paths with the sample input intothe fluidic system at block 1306, and mix with the sample. The mixtureof the sample and the second solution may then flow through the secondchannel and into the main detection channel where the sensor array islocated. The second solution may be a buffer solution. In one example,the second solution is a lysing buffer solution. The second solution maybe moved along and between the various channels using pressure drivenflow. The pressure may be caused by a syringe forcing liquid or airthrough the cartridge, or by pressurized air pushing against the secondsolution, to name a few examples. Other examples of techniques fortransporting the second solution through the cartridge includeelectro-wetting or using an on-chip peristaltic pump.

At block 1310, the biomolecules present within the sample are incubatedover the sensor array. Incubation may last for any given amount of time,for example, between 30 seconds and 10 minutes. During incubation, thesample mixed with the second solution may not be flowing, or may beflowing at a very slow flow rate. The flow rate may be designed suchthat fresh solution is presented over the sensor array over time, butthe flow is not too strong to cause damage to the capture reagents or tonot allow for the binding reactions to occur.

At block 1312, after the incubation time has expired, a third solutionflows through the first channel of the cartridge and through the maindetection channel to push substantially all of the sample mixed with thesecond solution into the waste chamber. The third solution may beinjected through the main detection channel for a given period of timeto ensure that the sample has been cleared from the main detectionchannel. The third solution used in block 1312 should ideally be thesame solution as the first solution. In another embodiment, the thirdsolution is different from the first solution. The third solution may bea buffer solution.

At block 1314, the output from the sensor array is measured to determineif any binding reactions occurred. The sensor output may be a draincurrent measured from one or more of the dual gate back-side sensing FETSensors in the sensor array. The measured drain current may be comparedto a drain current measured during calibration of the same sensor inblock 1304. If the threshold voltage (e.g., roughly corresponding to thevoltage needed to turn on the FET and cause the drain current to flow)has changed from when the sensor was calibrated, then it may bedetermined that a binding reaction has occurred and that a targetanalyte was present in the sample. The amount, and sign, of the changein threshold voltage may depend on numerous factors, such as whether thedual gate back-side sensing FET Sensor was an n-channel device or ap-channel device, the type of analyte being detected and the amount ofpositive or negative charge associated with the analyte. In anotherexample, the measured output from the sensor array is the thresholdvoltage itself, which may be compared to a threshold voltage measuredduring calibration of the same sensor in block 1304.

Chemistry, Biology and Interface

The apparatus, systems, and methods of the invention as described inthis application can be used to detect and/or monitor interactionsbetween various entities. These interactions include biological andchemical reactions to detect target analytes in a test sample. As anexample, reactions, including physical, chemical, biochemical, orbiological transformations, can be monitored to detect generation ofintermediates, byproducts, products, and combinations thereof. Inaddition the apparatus, systems, and methods of the invention can beused to detect these reactions in various assays as described herein,including, but not limited to, circulating tumor cell assays used inliquid biopsies and chelation assays to detect the presence of heavymetals and other environmental pollutants. Such assays and reactions canbe monitored in a single format or in an array format to detect, e.g.,multiple target analytes.

Biological Sensing Examples with DGBSS FET Sensor

Referring to FIG. 14, an example biosensing test is performed using thedual gate back-side sensing FET Sensor described above. Probe DNA 1404(an example of a capture reagent) is bound to interface layer 508 via alinking molecule 1402. Linking molecule 1402 may have a reactivechemical group that binds to a portion of interface layer 508. Anexample of linking molecules include thiols. Linking molecules may alsobe formed via silanization of the surface of interface layer 508, or byexposing the surface of interface layer 508 to ammonia (NH₃) plasma, inorder to form reactive NH₂ groups on the surface. The silanizationprocess involves sequentially exposing the surface of interface layer508 to different chemicals to build up covalently-bound molecules on thesurface of interface layer 508, as would be generally understood to aperson skilled in the relevant art. Probe DNA 1404 represent singlestranded DNA. According to an embodiment, linking molecule 1402 is boundto interface layer 508 before any steps of method 1300 are performed.Probe DNA 1404 may also be bound to linking molecule 1402 before anysteps of method 1300 are performed. In another example, probe DNA 1404is bound to linking molecule 1402 at block 1302 of method 1300.

The dual gate back-side sensing FET sensor illustrated in FIG. 14 is oneFET within a sensor array that would exist on a chip, such as chip 702described above, according to an embodiment. Linking molecule 1402 maybe bound to interface layer 508 before a wafer containing chip 702 isdiced to separate chip 702 from the wafer.

Probe DNA 1404 may be immobilized on interface layer 508 prior tosubjecting the FET Sensor to sample 1401. Sample 1401 may include thematching single stranded DNA sequence 1406 that binds strongly to itsmatching probe DNA 1404. The binding of additional DNA increases thenegative charge present on interface layer 508, and directly abovechannel region 208 of the FET Sensor.

The DNA binding is illustrated conceptually in FIG. 15A. Here probe DNAhaving nucleic acid sequence TCGA binds to its complementary matchedstrand having nucleic acid sequence AGCT. Any unmatched sequences willnot hybridize with the probe DNA sequences. The binding of the matchingDNA increases the negative charge built up at the interface of interfacelayer 508. In the example illustrated in FIG. 15A, interface layer 508is hafnium oxide.

FIG. 15B illustrates a shift in the threshold voltage of the dual gateback-side sensing FET Sensor when matching DNA is bound to the surfaceof interface layer 508. Briefly, voltage is applied to fluid gate 510until the FET Sensor “turns on” and current flows between drain region206 and source region 204. When more negative charge is present atinterface layer 508 due to complementary DNA binding, a higher voltageis required to form the conductive inversion layer within the channelregion 208. Thus, according to an embodiment, a higher voltage may beapplied to fluid gate 510 before the FET Sensor conducts and I_(ds)current flows. This difference in threshold voltage may be measured andused to determine not only the presence of the target matching DNAsequence, but also its concentration. It should be understood that a netpositive accumulated charge at interface layer 508 would cause thethreshold voltage to decrease rather than increase. Additionally, thechange in threshold voltage will have the opposite sign for an n-channelFET as compared to a p-channel FET.

Referring to FIG. 16, another example biosensing test is performed usingthe dual gate back-side sensing FET Sensor. Probe antibodies 1604(another example of capture reagents) are bound to interface layer 508via linking molecules 1602. Linking molecules 1602 may have a reactivechemical group that binds to a portion of interface layer 508. A samplesolution 1601 may be provided over probe antibodies 1604 to determine ifthe matching antigens are present within sample solution 1601. Accordingto an embodiment, linking molecules 1602 are bound to interface layer508 before any steps of method 1300 are performed. Probe antibodies 1604may also be bound to linking molecules 1602 before any steps of method1300 are performed. In another example, probe antibodies 1604 are boundto linking molecules 1602 at block 1302 of method 1300.

Referring to FIG. 17, the binding process of matching antigens to probeantibodies 1604 is illustrated. Here, matching antigens will bind to theimmobilized probe antibodies while unmatched antigens will not bind.Similar to the DNA hybridization process described above, the matchingantigens will change the accumulated charge present at interface layer508. The shift in threshold voltage due to the accumulated charge frommatching antibodies binding to the probe antibodies is measured insubstantially the same way as already discussed above with reference toFIG. 15B.

Final Remarks

It is to be appreciated that the Detailed Description section, and notthe Abstract of the Disclosure section, is intended to be used tointerpret the claims. The Abstract of the Disclosure section may setforth one or more but not all exemplary embodiments of the presentinvention as contemplated by the inventor(s), and thus, is not intendedto limit the present invention and the subjoined claims in any way.

It is to be understood that the phraseology or terminology herein is forthe purpose of description and not of limitation, such that theterminology or phraseology of the present specification is to beinterpreted by the skilled artisan in light of the teachings andguidance.

The breadth and scope of the present invention should not be limited byany of the above-described exemplary embodiments, but should be definedonly in accordance with the subjoined claims and their equivalents.

What is claimed is:
 1. An analyzer coupled with a fluidic cartridge, theanalyzer comprising: a syringe coupled to a fluidic channel of thefluidic cartridge; a needle aligned with the syringe and a fluid inletof the fluidic channel; an actuator configured to control the operationof the syringe; a sensing module coupled to a biological field effecttransistor (BioFET) sensor array of the fluidic cartridge, wherein thesensing module is configured to send and receive signals from the BioFETsensor array via a plurality of conductive pads on the fluidic cartridgewhen the fluidic cartridge is physically coupled to the analyzer; and aprocessor electrically coupled to the sensing module, wherein theprocessor is configured to determine a concentration level of a givenanalyte from a sample in the fluidic cartridge based on the signalsreceived from the BioFET sensor array.
 2. The analyzer of claim 1,wherein the syringe is detachable from the analyzer.
 3. The analyzer ofclaim 1, wherein the syringe is configured to hold and deliver a buffersolution to the fluidic channel.
 4. The analyzer of claim 1, wherein theneedle is configured to deliver a buffer solution to the fluidic channelthrough a plug disposed within the fluid inlet.
 5. The analyzer of claim1, wherein the syringe, the fluidic channel, and the fluid inlet extendin directions parallel to a substrate of the BioFET sensor array.
 6. Theanalyzer of claim 1, wherein the sensing module is configured toactivate the BioFET sensor array.
 7. The analyzer of claim 1, furthercomprising an actuator controller configured to control the operation ofthe actuator.
 8. The analyzer of claim 5, wherein the processor iselectrically coupled to the actuator controller.
 9. The analyzer ofclaim 1, further comprising a memory configured to store data related tothe concentration level of a given analyze determined by the processor.10. The analyzer of claim 1, further comprising a communications moduleconfigured to transmit data wirelessly or across a USB connection to anexternal processing device.
 11. The analyzer of claim 1, wherein theanalyzer and the fluidic cartridge are portable.
 12. A biosensoranalyzer, comprising: first and second syringes coupled to first andsecond fluidic channels of a fluidic cartridge; a first needle alignedwith the first syringe and a first fluid inlet of the first fluidicchannel; a second needle aligned with the second syringe and a secondfluid inlet of the second fluidic channel; first and second actuatorsconfigured to control the operation of the first and second syringes,respectively; a sensing module coupled to a biological field effecttransistor (BioFET) sensor array of the fluidic cartridge, wherein thesensing module is configured to activate the BioFET sensor array; and aprocessor electrically coupled to the sensing module, wherein theprocessor is configured to determine a concentration level of a givenanalyte from a sample in the fluidic cartridge based on the signalsreceived from the BioFET sensor array.
 13. The biosensor analyzer ofclaim 12, wherein the first and second syringes are configured todeliver buffer solutions to the BioFET sensor array through the firstand second fluidic channels.
 14. The biosensor analyzer of claim 12,wherein the first needle is configured to deliver a buffer solution tothe first fluidic channel through a first plug disposed within the firstfluid inlet.
 15. The biosensor analyzer of claim 12, wherein thebiosensor analyzer is portable.
 16. The biosensor analyzer of claim 12,wherein the first and second syringes are detachable from the biosensoranalyzer.
 17. A method of detecting an analyte in a sample, comprising:introducing a first solution through a first inlet into a first fluidicchannel to direct the first solution to a biological field effecttransistor (BioFET) sensor array; measuring a first signal from theBioFET sensor array in the first solution; introducing a sample to thefirst fluidic channel via a sample inlet coupled to the first fluidicchannel; introducing a second solution through a second inlet into asecond fluidic channel to mix with the sample and flow to the BioFETsensor array through a third fluidic channel; introducing a thirdsolution through the first inlet into the first fluidic channel to washthe sample away from the BioFET sensor array; measuring a second signalfrom the BioFET sensor array in the third solution; and comparing thefirst and second signals to determine the presence of an analyte in thesample.
 18. The method of claim 17, further comprising incubating thesample over the array of sensors for a given period of time beforeintroducing the third solution.
 19. The method of claim 17, wherein thefirst and third solutions are different from the second solution. 20.The method of claim 17, wherein the measuring of the first and secondsignals are performed by an analyzer coupled to a fluidic cartridge thatcomprises the first, second, and third fluidic channels and the BioFETsensor array.